Transformant for screening of inhibitors for human immunodeficiency virus

ABSTRACT

A microorganism cotransformed with a gene expressing HIV nucleocapsid protein and a plasmid vector containing HIV ψ gene and β-galactosidase reporter gene, and a method for screening HIV inhibitors employing the transformant. The invented method comprising the steps of culturing the transformant, treating it with putative compounds or compositions of HIV inhibitors, and measuring the degree of change in β-galactosidase expression in the culture, can be practically applied in screening HIV packaging inhibitors by which the interaction between HIV nucleocapsid and HIV ψ sequence is blocked.

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/KR00/01173, filed Oct. 18, 2000, theentire contents of which are incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a transformant for screening of humanimmunodeficiency virus(“HIV”) inhibitors, more particularly, to atransformant cotransformed with a plasmid expressing HIV nucleocapsidprotein and a plasmid containing HIV psi(ψ) nucleotide sequence andβ-galactosidase reporter gene, and a method for screening of HIVinhibitors by employing the said transformant.

2. Background of the Invention

HIV, a pathogen causing the aquired immunodeficiency syndrome (“AIDS”),selectively infects crucial immune cells called CD4+ T helper cells andreplicates inside the cells. Infection of HIV leads to the lysis of CD4+T cells resulting from an interaction between viral env glycoprotein andplasma membrane of target cell and a subsequent reproduction of virusparticles. Also, the binding of soluble gp120 to CD4+ molecules ontouninfected T cells block interactions of CD4+ T cells with other immunecells. In addition to depleting CD4+ T cells, impaired are function ofcytotoxic T cells expressing CD8+, antibody-dependent cytotoxicity,maturation of CD4+ T cells in thymus, interaction between CD4+ cells andclass II MHC on antigen presenting cells, and function of macrophagesand natural killer cells. Thus, human immune system is graduallydeteriorated after HIV infection.

Until now, drugs suppressing HIV replication have been developed, whichinclude reverse transcriptase inhibitors such as AZT(azidothymidine) andddI(dideoxyinosine), and protease inhibitors. Recently, the researchesto develop DNA vaccines employing nucleotide sequence encoding HIVproteins (see: Hinkula J. et al., Vaccine, 15:874–878, 1997; Calarota etal., Lancet, 351:1320–1325, 1998), and live-attenuated HIV vaccines madeby deleting the HIV nef gene are being undertaken (see: Kestler andJeang, Science, 270:1219–1222, 1995; Chakrabarti et al., Proc. Natl.Acad. Sci., USA, 93:9810–9815, 1996).

Since the said drugs are not able to remove the provirus of HIV of whichDNA is inserted into the host immune cell chromosome or not able toselectively remove host immune cells containing the provirus, it cannotbe excluded that HIV variants arisen by genetic mutation acquire drugresistance or HIV revertants arisen by recombination of attenuated virusvaccine acquire characteristics of pathogenic HIV (see: Berkhout et al.,J. Virol., 73:1138–1145, 1999). Furthermore, it has been found that HIVrequires not only CD4+ molecule as the receptor on the surface of hostcells, but also coreceptors such as ‘T-cell-line-tropic’ CXCR4/fusincoreceptor or ‘macrophage-tropic’ CCR5 coreceptor for its binding andgaining entry of HIV into host cells (see: Feng et al., Science,272:872–877, 1996). Thus, one approach for drug therapy is to targetthese coreceptors in an attempt to inhibit binding of virus onto thehost cells. Since the normal function of these coreceptors is to bind‘chemokines (chemotactic cytokines)’ which plays a role in inflammationreaction, serious side effects may be anticipated (see: Murphy, P. M.,Ann. Rev. Immunol., 12:593–633, 1994). In view of above situation, thereis a need to develop a novel class of HIV inhibitors which do not affecthost immunity or physiological activity relating to receptors, and oneapproach for such drug therapy is to target HIV specific factorsrequired for virus assembly.

When HIV virus particle is assembled, its genomic RNA is selectivelypackaged into a virion. It is well known that a specific interactionbetween the region of nucleocapsid (NC) protein and the viral packagingsequence (encapsidation signal), psi(ψ), allows selective packaging ofviral genomic RNA. Psi(ψ), located between long terminal repeat(LTR) of5′-terminal of genomic RNA and gag gene which encodes precursor polyprotein (matrix-capsid-nucleocapsid), has 4 stem-loop structures.However, the screening of compounds or compositions which inhibit thespecific viral interaction required for packaging is difficult due tothe lack of an easy and efficient assay system for such screening.

Under the circumstances, there are strong reasons for exploring anddeveloping a model system which can be used to detect the specificinteraction between HIV NC protein and HIV psi(ψ) sequence as in invivo, for screening of inhibitors against HIV packaging.

SUMMARY OF THE INVENTION

The present inventors have made an effort to develop a simple andeffective method for detecting the specific interaction between HIV NCprotein and HIV psi(ψ) sequence in vivo to screen HIV packaginginhibitors, thus, prepared transformants cotransformed with a plasmidexpressing HIV NC protein and a plasmid containing HIV psi(ψ) sequenceand β-galactosidase reporter gene, and found that HIV inhibitors can beconveniently screened by employing the said transformant on the basis ofthe expression level change of β-galactosidase.

A primary object of the invention is, therefore, to provide atransformant cotransformed with a plasmid expressing HIV nucleocapsidprotein, and a plasmid containing HIV psi (ψ) sequence andβ-galactosidase reporter gene.

The other object of the invention is to provide a method for screeningHIV packaging inhibitors employing the said transformant.

BRIEF DESCRIPTION OF THE DRAWINGS

The above, the other objects and features of the invention will becomeapparent from the following descriptions given in conjunction with theaccompanying drawings, in which:

FIG. 1 a is a schematic representation of construction strategy ofpX1(−ATG).

FIG. 1 b is a schematic representation of construction strategy of pNH1.

FIG. 1 c is a schematic representation of construction strategy ofpNH1Psi or pNH1rePsi.

FIG. 2 is a graph showing the expression of β-galactosidase in E. coliJM109 cotransformed with each of pJC1 expressing nucleocapsid protein,or pTrcHisGag expressing Gag protein, or pSE380 as a control plasmid,and pNH1, respectively.

FIG. 3 a is a graph showing the effect of interaction between HIVnucleocapsid protein or Gag protein and HIV psi(ψ) sequence on theexpression of β-galactosidase after induction with IPTG in E. coli JM109cotransformed with each of pJC1, pTrcHisGag or pSE380 and pNH1MCS,respectively.

FIG. 3 b is a graph showing the effect of specific interaction betweenHIV nucleocapsid protein or Gag protein and HIV psi(ψ) sequence on theexpression of β-galactosidase after induction with IPTG in E. coli JM109cotransformed with each of pJC1, pTrcHisGag or pSE380 andpNH1Psi(SL1234), respectively.

FIG. 4 is a graph showing the effect of specific interaction between HIVnucleocapsid protein or Gag protein and various portions of psi(ψ)sequence on the expression of β-galactosidase in E. coli JM109 which iscotransformed with each of pJC1, pTrcHisGag or pSE380 and each ofplasmid pNH1Psi(SL1234), pNH1Psi(SL234), pNH1Psi(SL34), pNH1Psi(SL23),or pNH1Psi(SL12) containing psi(ψ) nucleotide sequence, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The invented transformant employed for screening of HIV packaginginhibitors is prepared by cotransforming a plasmid pJC1 expressing HIVNC protein and a plasmid pNH1Psi(SL1234) containing HIV psi(ψ) gene andβ-galactosidase reporter gene.

The process for preparing the transformant for screening of HIVinhibitors is further illustrated in more detail.

The transformant was prepared by cotransforming with pJC1 expressing HIVNC protein and pNH1Psi(SL1234) containing HIV psi(ψ) gene andβ-galactosidase reporter gene: pX1(−ATG) was constructed by removing ATGfrom pX1 plasmid which was made by inserting lacZ gene fragment intopSE280. And then, pZ1 was obtained by substituting ampicillin resistancegene of pX1(−ATG) with the AflIII-StuI fragment containing kanamycinresistance gene. pNH1 containing lacZ gene was constructed by insertingHindIII-BspH1 fragment containing rrnB T1T2 terminator from pX1(−ATG)into pZ1. pNH1Psi(SL1234) containing lacZ gene which is flanked by HIVpsi(ψ) nucleotide sequence right before the starting codon wasconstructed by inserting 4 stem-loop structure-containing psi(ψ)fragment from pLLIII into pNH1 constructed above (see: FIG. 1). Andthen, NC protein-expressing pJC1 vector which is constructed by thepresent inventors (see: Ji Chang You and Charles S. McHenry, J. Biol.Chem., 268:16519–16527, 1993) and pNH1Psi(SL1234) constructed above arecotransformed into E. coli. That is, vectors pJC1 and pNH1Psi(SL1234)are cotransformed into E. coli by electroporation, followed by selectingantibiotics resistant transformant in medium containing antibiotics. Thetransformant was named E. coli/pNH1Psi(SL1234), and deposited with aninternational depository authority, the Korean Culture Center ofMicroorganisms (KCCM, #361–221 Hongje-1-dong, Seodaemun-gu, Seoul,Republic of Korea), under an accession (deposition) No. KCCM-10194 onMar. 31, 2000. The transformant prepared above has a characteristic thatβ-galactosidase reporter gene expression is down regulated due to theinteraction of psi(ψ) nucleotide sequence with HIV NC protein, therebyit can be used for screening of HIV packaging inhibitors. For instance,the culture of said transformant is treated with putative compounds orcompositions of HIV inhibitors, and then the expression level change ofβ-galactosidase in culture is determined. Therefore, the transformant ofthe invention can be used for screening of HIV packaging inhibitorswhich block the binding of HIV NC protein to HIV psi(ψ) nucleotidesequence.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

EXAMPLE 1 Construction of pNH1(SL1234)

A plasmid containing β-galactosidase reporter gene (SEQ ID NO: 1) wasconstructed.

EXAMPLE 1-1 Construction of pNH1

LacZ gene fragment obtained by PstI (Boeringer Mannheim, Germany)digestion of pUS935(see: Dellagostin, O. A. et al., Microbiology,141:1785–1792, 1995) was inserted into pSE280(Invitrogen, U.S.A.) toobtain pX1. In order to prevent translation from starting at ATG whichis located upstream of the multicloning site of LacZ fragment, upstreamATG was removed by cutting pX1 with NcoI, mung bean exonuclease(Boehringer Mannheim, Germany) treatment, Klenow enzyme treatment tocreate blunt end and then ligating to make pX1(−ATG). FIG. 1 a is aschematic representation of construction strategy of pX1(−ATG). pZ1 wasmade by substituting ampicillin resistance gene of pX1(−ATG) with akanamycin resistance gene, that is, by ligating the lacZ gene-containingMscI fragment from pX1(−ATG) and Kan^(r) gene-containing AflIII-StuIfragment from pZerO-2(Invitrogen, U.S.A.). Then, pNH1 was made byinserting rrn T1T2 terminator-containing HindIII-BspHI fragment frompX1(−ATG) into the ScaI site of pZ1. FIG. 1 b is a schematicrepresentation of construction strategy of pNH1.

EXAMPLE 1-2 Construction of pNH1Psi(SL1234)

pLLIII (University of Colorado, Health Sciences Center, Charles S.McHenry), a plasmid containing the 5′ long terminal rep eat (LTR) ofHIV-1, was digested with SacI and MseI and then treated with Klenowenzyme to obtain a fragment containing HIV psi(ψ) nucleotide sequence(named “SL1234”, SEQ ID NO: 2) which contains 4 stem-loop structures.

SL1234 was digested with MaeI and then treated with Klenow fragment toobtain SL12(SEQ ID NO: 3). pNH1Psi(SL1234) or pNH1Psi(SL12) was made byinserting SL1234 or two fragments of SL12 into the BstEII site which islocated upstream of lacZ gene in pNH1, respectively. Then,pNH1rePsi(SL1234) or pNH1rePsi(SL12) containing HIV psi(ψ) nucleotidesequence in reverse orientation was made to be used as a control vector.DNA bands of 221 bp for right orientation and 152 bp for reverseorientation were identified by 2% agarose gel electrophoresis.

Primers, U2 and L3 pair or U2 and L4 pair, were used for amplificationof SL23(SEQ ID NO: 4) or SL234(SEQ ID NO: 5), respectively, and theirnucleotide sequences are as follows.

-   -   primer U2 (SEQ ID NO: 6) 5′-GGGGGTGACCTTTAAAAGCAAGAGGCGAGGG-3′    -   primer L3 (SEQ ID NO: 7) 5′-GGGGGTGACCCTCTCCTTCTAGCCTCCG-3′    -   primer L4 (SEQ ID NO: 8) 5′-GGGGGTGACCGACGCTCTCGCACCCGTCTCT-3′

In order for easier insertion of psi(ψ) fragment into BstEI site inpNH1, BstEII site (boldface) was inserted into the each primer, and DraIsite (boldface) was inserted into the primer U2 to identify orientation.Also, point mutation (underlined), T to G, was introduced into primer L4to prevent translation from starting in the psi(ψ) sequence. PCR wasperformed in a 100 μl of reaction mixture containing 10 mM Tris-HCl, pH7.5, 1.5 mM MgCl₂, 50 mM KCl, 1 mM dNTP, 10 M of each primer, 1 μg ofpLLIII and 2.5 unit of Taq polymerase (Boehringer Mannheim, Germany)with preincubation at 94° C. for 5 min, 30 cycles of denaturation at 94°C. for 1 min, primer annealing at 60° C. for 2 min, and extension at 72°C. for 2 min, and then additional extension at 72° C. for 10 min. ForSL23 insertion, DNA bands of 213 bp for right orientation and 146 bp forreversed orientation were identified by 2% agarose gel electrophoresisfollowing digestion of each plasmid with DraI. For SL234 insertion, DNAbands of 234 bp for right orientation and 146 bp for reversedorientation were identified by 2% agarose gel electrophoresis followingdigestion of each plasmid with DraI.

SL34 fragment obtained by digestion of PCR product, SL234, with RsaI andBstEII, was treated with Klenow fragment, and then inserted into theBstEII site of pNH1 to make pNH1Psi(SL34) and pNH1rePsi(SL34) as acontrol. DNA bands of 40 bp for right orientation and 56 bp for reversedorientation were identified by 4% agarose gel electrophoresis followingdouble-digestion of each plasmid with BsmAI and XmnI.

In order to use as a negative control, that is, to see whether NCprotein or Gag protein can interact with nucleotide sequence which isnon-homologous to psi(ψ), pNH1MCS was made by inserting 75 bp MCS(multicloning site) fragment, which has no ATG starting codon, obtainedby digestion of pSE280 with MaeI and subsequent Klenow treatment, intothe BstEII site of pNH1. DNA band of 229 bp was identified by 2% agarosegel electrophoresis following RsaI digestion. FIG. 1 c is a schematicrepresentation of construction strategy of pNH1Psi or pNH1rePsi.

EXAMPLE 2 Expression of HIV NC Protein or HIV Gag Protein

NC protein-expressing plasmid, pJC1, which was made and disclosed by thepresent inventors (see: Ji Chang You, and Charles S. McHenry, J. Biol.Chem., 268(22):16519–16527, 1993) or Gag protein-expressing plasmid,pTrcHisGag, was transformed into E. coli strain JM109(Promega Co.,U.S.A.) by electroporation: to obtain competent cells, overnight cultureof JM109 started from single colony was inoculated into 200 ml of freshLB medium (Luria-Bertani, Difco, U.S.A.) with 1:100 dilution andcultivated to OD 0.5–0.7, and then cells were centrifuged at 4000 g for10 min., followed by 4 times of washing process which was resuspendingcell pellet in cold 10% (v/v) glycerol and centrifugation at 4000 g for10 min. After being resuspended in 1 ml, 20 μl of competent cells weremixed with 1–100 ng of DNA, transferred to 0.2 cm gap electroporationcuvette, and transformed in E. coli pulser (Bio-Lab, Heracules, Calif.,U.S.A.) by applying 2.5 kV electric shock. The transformant wasincubated overnight in LB medium containing 100 μg of ampicillin, andthen subcultured in fresh LB medium with 1:100 dilution. At the earlyperiod of logarithmic phase of growth, 1 mM ofisopropyl-β-D-thiogalactopyranoside (IPTG, Roche Diagnostics, Germany)was added and the proteins were induced for 3 hours. The liquid cultureof transformant was sampled at each hour and centrifuged at 10,000 g for1 min. The cell pellet was resuspended in 20 μl of gel loading buffer(50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenolblue and 10% glycerol), and incubated for 5 min at 100° C. to disruptcell wall. After centrifugation at 10,000 g for 30 min, each supernatantwas subject to 15% SDS-PAGE at 120V for 2 hours. The gel was stained instaining solution (0.25% Coomassie Brilliant Blue, 45% methanol and 10%glacial acetic acid) for 20 min and then destained in destainingsolution (30% methanol and 10% acetic acid) for 2 hours. Although HIV NCprotein (7 kD) band did not appear without IPTG induction, the proteinband at 7 kD position was getting thicker after IPTG induction as timewent by Also, HIV Gag protein (55 kD) band appeared at about 55 kDposition after IPTG induction.

EXAMPLE 3 Cotransformation and Measurement of β-galactosidase expression

In order to measure the effect of interaction between HIV psi(ψ)nucleotide sequence and HIV NC protein or Gag protein on β-galactosidaseexpression level, pJC1 and each of pNH1Psi plasmids or pTrcHisGag andeach of pNH1Psi plasmids were cotransformed into E. coli JM109 byelectroporation, respectively. To exclude the possibility of plasmidcopy number effect and to confirm the expression of lac repressor, JM109was cotransformed with pSE380(Invitrogen, U.S.A.) as a control plasmidand each of pNH1Psi plasmids. Each cotransformant was selected on LBagar plate containing 100 μg/ml ampicillin and 40 μg/ml kanamycin.

To measure the expression of β-galactosidase in the cotransformants,β-galactosidase liquid assay was performed for each cotransformant morethan 3 times. Each cotransformant was cultured in LB medium overnight,and the culture broth was inoculated into 5 ml of fresh LB medium with1:10 dilution. At the early stage of logarithmic phase of growth, 1 mlaliquot of culture broth was taken and kept on ice, and then incubationwas continued for 3 hours to allow expression of NC protein, Gag proteinand β-galactosidase after 1 mM IPTG was added for induction ofβ-galactosidase. And then, 1 ml aliquot of culture broth was sampledeach hour and kept on ice. Cell density was measured at A₆₀₀ withenzyme-linked immunosorbent assay reader (ELISA reader, Dynatech,U.S.A.). For measurement of β-galactosidase activity, 50 g aliquot ofeach cotransformant culture broth was mixed with 450 μl aliquot of Zbuffer (60 mM Na₂HPO₄ 7H₂O, 40 mM NaH₂PO₄.H₂O, 10 mM KCl, 1 mM MgSO₄7H₂O, 50 mM β-mercaptoethanol). And then, cell wall was disrupted byaddition of 20 μl of chloroform and 10 μg of 0.1% SDS followed bystirring for 30 seconds and incubating at 28° C. for 5 min. To the celllysate, 100 μl of o-nitrophenyl-1-thio-β-D-galactopyranoside (ONPG, 4mg/ml, Sigma, U.S.A.) was added and the mixture was incubated at roomtemperature until yellow color developed. When yellow color was fullydeveloped, the reaction was stopped by adding 200 μl of 1M Na₂CO₃, andthe mixture was clarified by centrifugation at 10,000 g for 30 min.Optical density (OD) of supernatant was measured at 420 nm and 550 nm.The activity (units) of β-galactosidase was quantitated by followingequation:

Enzyme  unit  of  β-galactosidase = 1, 000 × (OD₄₂₀ − 1.75 × OD₅₅₀)/(t × OD₆₀₀)

wherein,

-   -   t is reaction time (minutes); and,    -   1.75 is a constant.

In order to determine if NC protein or Gag protein affect theβ-galactosidase expression from the plasmid without psi(ψ) sequence, E.coli JM109 was cotransformed with the precursor plasmid, pNH1(containinglacZ gene, no psi(ψ) sequence) and pJC1(nucleocapsid), pNH1 andpTrcHisGag (Gag protein), or pNH1 and pSE380(control), respectively, andthen, β-galactosidase activity was measured as described above. FIG. 2is a graph showing the expression of β-galactosidase in JM109cotransformed with each of pJC1 expressing nucleocapsid protein,pTrcHisTag expressing Gag protein, or pSE380 as a control plasmid, andpNH1. As shown in FIG. 2, the level of β-galactosidase expression incotransformant expressing NC protein or Gag protein was not differentfrom that in cotransformant expressing no other protein (pSE380) after 3hour-induction of β-galactosidase with IPTG, therefore, it wasdemonstrated that NC protein or Gag protein had no effect on theexpression of β-galactosidase in cotransformants.

In order to demonstrate the specificity of the interaction between HIVNC protein and HIV psi(ψ) sequence or Gag protein and HIV psi(ψ)sequence, β-galactosidase expression was measured in cotransformantscontaining each of pSE380, pJC1 or pTrcHisGag and each of pNH1MCS(multiple cloning site, MCS, as a non-homologous sequence to HIV psi(ψ)nucleotide sequence) or pNH1Psi(SL1234, HIV psi(ψ) nucleotide sequence),respectively. FIG. 3 a is a graph showing the effect of interactionbetween HIV nucleocapsid protein or Gag protein and non-homologoussequence to psi(ψ) on the expression of β-galactosidase after inductionwith IPTG in JM109 cotransformed with each of pJC1, pTrcHisTag or pSE380and pNH1MCS. FIG. 3 b is a graph showing the effect of specificinteraction between HIV nucleocapsid protein or Gag protein andhomologous psi(ψ) sequence on the expression of β-galactosidase afterinduction with IPTG in JM109 cotransformed with each of pJC1, pTrcHisGagor pSE380 and pNH1Psi(SL1234), respectively. As shown in FIG. 3 a, thelevel of β-galactosidase in cotransformants containing non-homologoussequence (pNH1MCS) to psi(ψ) and a plasmid expressing NC protein or Gagprotein was not different from that in cotransformant containing pHN1MCSand control plasmid, pSE380. Meanwhile, as shown in FIG. 3 b, whentransformed together with pNH1psi(SL1234), the level of β-galactosidasein cotransformant expressing Gag protein was reduced by 15% compared tothat in cotransformant containing control vector, pSE380, moreover,cotransformant expressing NC protein showed more than 90% reduction inβ-galactosidase expression compared to the control cotransformant.

Therefore, it was demonstrated that β-galactosidase expression incotransformant was reduced by HIV NC protein in the presence of HIVpsi(ψ) nucleotide sequence and the reduction was caused by a specificinteraction between HIV NC protein and HIV psi(ψ) nucleotide sequence.

In order to identify which portion of psi(ψ) nucleotide sequence isresponsible for the specific interaction with NC protein, each ofplasmids containing various portions of stem-loop structures of psi (ψ)nucleotide sequence were cotransformed with NC- or Gag-expressing vectorinto E. coli JM109, respectively, and then β-galactosidase activity ofeach cotransformant was measured. FIG. 4 is a graph showing the effectof specific interaction between HIV NC protein or Gag protein andportions of psi(ψ) sequence on the expression of β-galactosidase in E.coli JM109 cotransformants. E. coli JM109 was cotransformed with each ofpJC1, pTrcHisGag, or pSE380 and each of plasmids containing variousportion of stem-loop structures of psi(ψ) nucleotide sequence,pNH1Psi(SL1234), pNH1Psi(SL234), pNH1Psi(SL34), pNH1Psi(SL23), orpNH1Psi(SL12), respectively. In FIG. 4, horizontal axis indicates thecotransformants containing each of pJC1, pTrcHisGag, or pSE380 and eachof psi(ψ) structure-containing plasmids (lane 1 is control plasmid,pNH1; lane 2, a plasmid containing non-homologous sequence, pNH1MCS;lane 3, pNH1Psi(SL1234); lane 4, pNH1Psi(SL234); lane 5, pNH1Psi(SL34);lane 6, pNH1Psi(SL23); and, lane 7, pNH1Psi(SL12)), and vertical axisindicates the activity of β-galactosidase. The results werestatistically analyzed by Student's test and * indicates p<0.05. Asshown in FIG. 4, HIV NC protein reduced the expression of lacZ gene moreeffectively when psi(ψ) sequence is present in cell than HIV Gag proteindid. When pNH1Psi(SL1234) containing full length psi(ψ) nucleotidesequence and pJC1 were cotransformed into pJM109, the reduction of lacZexpression was most significant, and the degree of reduction wasdecreased in the order of SL34, SL234, SL23, and SL12. The sequence ofSL4 appears to be more important than other stemp-loop structures forthe specific interaction of psi(ψ) sequence with NC protein whichresulted in the reduction of lacZ gene expression.

Therefore, these results demonstrated that all of 4 stem-loop structuresin psi(ψ) sequence was required for the most significant reduction ofβ-galactosidase expression, and sequence of SL4 was the most importantportion in psi(ψ) sequence for NC protein binding. And, the degree ofreduction of lacZ gene expression in pNH1Psi(SL234) and pNH1Psi(SL34)which were point-mutated to avoid translation starting from SL4 area wassimilar to that in pNH1Psi(SL1234) without point mutation. Thus, thetransformant was named as E. coli/pNH1Psi(SL1234), which showed the mostsignificant reduction of β-galactosidase expression by specificinteraction of psi(ψ) sequence with NC protein. E. coli/pNH1Psi(SL1234)was deposited with an international depository authority, the KoreanCulture Center of Microorganisms (KCCM, #361–221 Hongje-1-dong,Seodaemun-gu, Seoul, Republic of Korea), under an accession (deposition)No. KCCM-10194 on Mar. 31, 2000. The results obtained from the aboveExamples were summarized in Table 1 below.

TABLE 1 Level of β-galactosidase expression depending on kinds ofreporter vectors Enzyme units of β-galactosidase after 3-hour inductionpJC1 pTrcHisGag pSE380 (expressing (expressing Reporter Vector (Control)NCp7) Gag) pNH1 — 302 ± 19* 100% 295 ± 28.7 98% 298 ± 20.8 96% pNH1MCS75 bp 296 ± 24.5 100% 234 ± 40.5 80% 296 ± 44.5 100%  pNH1Psi SL1234 119± 22 100%  8 ± 2.2 6.7% 103 ± 23 86.5% (SL1234) pNH1Psi SL234 308 ± 42.5100%  87 ± 25.5 28% 140 ± 44 76% (SL234) pNH1Psi SL34  85 ± 14 100%  11± 2.6 13%  68 ± 12 80% (SL34) pNH1Psi SL23 298 ± 55.5 100% 112 ± 14 38%214 ± 22.5 72% (SL23) pNH1rePsi SL12 186 ± 32 100% 107 ± 3.5 58% 158 ±24 85% (SL12) pNH1rePsi reSL1234  87 ± 21 100%  51 ± 10 59%  85 ± 17 98%(SL1234) pNH1rePsi reSL234 185 ± 37 100%  78 ± 5 42% 140 ± 30.5 76%(SL234) pNH1rePsi reSL34 311 ± 29 100% 186 ± 60% 307 ± 30 99% (SL34)pNH1rePsi reSL23 305 ± 43 100% 131 ± 19 43% 280 ± 18 92% (SL23) PNH1PsireSL12  86 ± 21 100%  64 ± 11 75%  86 ± 19.5 100%  (SL12)

As a result, it is a characteristic of the transformant to be reduced inβ-galactosidase reporter gene expression due to the specific interactionbetween psi(ψ) nucleotide sequence and HIV NC protein. Therefore, thetransformant of the invention can be used for screening of HIV packaginginhibitors which block the binding of HIV NC protein to HIV psi(ψ)nucleotide sequence, by treating the culture broth of E. coliJM109(KCCM-10194) with putative compounds or compositions of HIVinhibitors and measuring the degree of change in β-galactosidaseexpression.

As clearly illustrated and demonstrated above, the present inventionprovides a microorganism cotransformed with a gene expressing HIVnucleocapsid protein and a plasmid vector containing HIV Psi (ψ) geneand β-galactosidase reporter gene, and a method for screening HIVpackaging inhibitors employing the said transformant. The inventedmethod comprising the steps of culturing the said transformant, treatingit with putative compounds or compositions of HIV inhibitors, andmeasuring the degree of change in β-galactosidase expression in theculture, can be practically applied in screening HIV packaginginhibitors by which the interaction between HIV nucleocapsid and HIVpsi(ψ) sequence is blocked.

1. A microorganism cotransformed with a plasmid vector containing a geneexpressing the HIV nucleocapsid protein, and a plasmid vector containingthe HIV psi (ψ) sequence and a reporter gene located downstream of theHIV psi (ψ) sequence, wherein reporter gene expression is downregulatedby the specific binding interaction of the psi sequence with thenucleocapsid protein.
 2. The microorganism of claim 1 wherein theplasmid vector containing a gene expressing the HIV nucleocapsid proteinis pJC1.
 3. The microorganism of claim 1 wherein the HIV psi (ψ)sequence is selected from the group consisting of SL1234 (SEQ ID NO: 2),SL234 (SEQ ID NO: 5), SL23 (SEQ ID NO: 4), and SL12 (SEQ ID NO: 3).
 4. Amicroorganism comprising E. coli JM109 (KCCM-10194) cotransformed with avector pJC1 expressing the HIV nucleocapsid protein, and a vectorpNH1Psi(SL1234) containing the HIV psi(ψ) sequence and β-galactosidasereporter gene (SEQ ID NO: 1) located downstream of the HIV psi(ψ)sequence, wherein β-galactosidase expression is downregulated by thespecific binding interaction of the psi sequence with the nucleocapsidprotein.
 5. A microorganism cotransformed with the vector pJC1expressing the HIV nucleocapsid protein, and a vector pNH1Psi(SL234)containing the HIV psi (ψ) sequence and β-galactosidase reporter gene(SEQ ID NO: 1) located downstream of the HIV psi(ψ) sequence, whereinβ-galactosidase expression is downregulated by the specific bindinginteraction of the psi sequence with the nucleocapsid protein.
 6. Amicroorganism cotransformed with the vector pJC1 expressing the HIVnucleocapsid protein, and a vector pNH1Psi(SL23) containing the HIV psi(ψ) sequence and β-galactosidase reporter gene (SEQ ID NO: 1) locateddownstream of the HIV psi(ψ) sequence, wherein β-galactosidaseexpression is downregulated by the specific binding interaction of thepsi sequence with the nucleocapsid protein.
 7. A microorganismcotransformed with the vector pJC1 expressing the HIV nucleocapsidprotein, and a vector pNH1Psi(SL12) containing the HIV psi(ψ) sequenceand β-galactosidase reporter gene (SEQ ID NO: 1) located downstream ofthe HIV psi(ψ) sequence, wherein β-galactosidase expression isdownregulated by the specific binding interaction of the psi sequencewith the nucleocapsid protein.
 8. A microorganism transformed with avector pNH1Psi(SL1234) containing the HIV psi (ψ) gene andβ-galactosidase reporter sequence (SEQ ID NO: 1) located downstream ofthe HIV psi(ψ) sequence, wherein β-galactosidase expression isdown-regulated by the specific binding interaction of the psi sequencewith the nucleocapsid protein.
 9. A microorganism wherein both a plasmidvector containing a gene coding for the HIV nucleocapsid protein and aplasmid vector containing the HIV psi (ψ) sequence and β-galactosidasereporter gene (SEQ ID NO: 1) located downstream of the HIV psi(ψ)sequence are integrated into a chromosome, wherein β-galactosidaseexpression is downregulated by the specific binding interaction of thepsi sequence with the nucleocapsid protein.
 10. A method of screeningfor HIV packaging inhibitors which comprises the steps of: (i) culturingthe cotransformed microorganism of claim 1; (ii) treating the saidcotransformed microorganism with putative compounds or compositions ofHIV inhibitors; and, (iii) measuring the degree of change in reportergene expression in the culture, wherein an increase in reporter geneexpression in the presence of the compound or composition compared toreporter gene expression in the absence of the compound or compositionindicates the compound or composition inhibits the specific bindinginteraction between the HIV nucleocapsid protein and the psi sequence.11. The method of claim 10 wherein the cotransformed microorganism is E.coli JM109 (KCCM-10194).
 12. The microorganism of claim 1, wherein thereporter gene is β-galactosidase.
 13. The microorganism of claim 12wherein the β-galactosidase reporter gene is SEQ ID NO:
 1. 14. Themicroorganism of claim 13 wherein the plasmid vector containing the HIVpsi(ψ) sequence and β-galactosidase reporter gene is selected from thegroup consisting of pNH1Psi(SL1234), pNH1Psi(SL234), pNH1Psi(SL23),pNH1Psi(SL12), and pNH1Psi(SL34).